This invention is directed to methods for detecting polymorphisms in complex eukaryotic genes, particularly the gene for ataxia telangiectasia, and to polymorphisms detected by those methods.
Many autosomal recessive genetic disorders are caused by mutations in complex single genes that cause the genes to malfunction, producing a defective product or no product at all. Many of these genes include multiple exons, promoters, and other significant regions.
Ataxia-telangiectasia (A-T) (MIM208900) is an autosomal recessive disorder characterized by progressive cerebellar degeneration, immunodeficiency, growth retardation, premature aging, chromosomal instability, acute sensitivity to ionizing radiation, and cancer predisposition (R. A. Gatti, xe2x80x9cAtaxia-Telangiectasiaxe2x80x9d in Genetic Basis of Human Cancer (Vogelstein and Kinzler, eds. McGraw-Hill, N.Y., 1998)).
The gene responsible for A-T, ATM, was initially localized to chromosome 11q23.1 (E. Lange et al., xe2x80x9cLocation of an Ataxia-Telangiectasia to a xcx9c500 kb Interval on Chromosome 11q23.1:Linkage Analysis of 176 Families in an International Consortium,xe2x80x9d Am. J. Hum. Genet. 57:112-119 (1995); N. Uhrhammer et al., xe2x80x9cSublocalization of an Ataxia-Telangiectasia Gene Distal to D11 S384 by Ancestral Haplotyping in Costa Rican Families,xe2x80x9d Am. J. Hum. Genet. 57:103-111 (1995)) and, on this basis, was positionally cloned by Savitsky et al. (K. Savitsky et al., xe2x80x9cA Single Ataxia-Telangiectasia Gene with a Product Similar to a PI-3 Kinase,xe2x80x9d Science 268:1749-1753 (1995)). It spans about 150 kb of genomic DNA, encodes a major transcript of 13 kb, and a 370 kDa protein (G. Chen and E. Y. H. P. Lee, xe2x80x9cThe Product of the ATM Gene is a 370-kDa Nuclear Phosphoprotein,xe2x80x9d J. Biol. Chem. 271:33693-33697 (1996)). Subsequently, a wide spectrum of ATM mutations has been detected in A-T patients, spread throughout the gene and without evidence of a mutational hot spot (P. Concannon and R. A. Gatti, xe2x80x9cDiversity of ATM Gene Mutations in Patients with Ataxia-Telangiectasia,xe2x80x9d Hum. Mutat. 10:100-107 (1997)).
Procedures used for mutation screening in the ATM gene have included restriction-endonuclease fingerprinting (REF) (K. Savitsky et al. supra (1995); P. J. Byrd et al., xe2x80x9cMutations Revealed by Sequencing the 5xe2x80x2 Half of the Gene for Ataxia-Telangiectasia,xe2x80x9d Hum. Mol. Genet. 5:145-149 (1996)), the single-strand conformation polymorphism (SSCP Technique) J. Wright et al., xe2x80x9cA High Frequency of Distinct ATM Mutations in Ataxia in Telangiectasia,xe2x80x9d Am. J. Hum. Genet. 59:839-846 (1996); T. Sasaki et al., xe2x80x9cATM Mutations in Patients with Ataxia-Telangiectasia Screened by a Hierarchical Strategy,xe2x80x9d Hum. Mutat. 12:186-195 (1998)), and the protein truncation test (PTT); (M. Telatar et al., xe2x80x9cAtaxia-Telangiectasia: Mutations in ATM cDNA Detected by Protein-Truncation Screening,xe2x80x9d Am. J. Hum. Genet. 59:40-44 (1996)).
The ATM gene shows homology with protein kinases in yeast (TEL-1), drosophila (Mei-41) and human (DNA-PK) and is most closely related to DNA-PK and TEL-1(Savitsky et al., (1995), supra; K. Savitsky et al., Hum. Mol. Genet. 4:2025-2032 (1 995); Lehmann et al., Trends Genet. 11:375-377 (1995); Zakin, Cell 82:685-687 (1995); Lavin et al., Trends Biol. Sci. 20:382-383 (1995); Keith et al., Science 270:50-51 (1995)).
The nucleotide sequence encoding the ATM protein is SEQ ID NO: 1. This corresponds to GenBank Accession No. U33841. The open reading frame is 9168 nucleotides. There is a 3xe2x80x2 untranslated region (UTR) and a 5xe2x80x2 UTR. SEQ ID NO: 2 is the amino acid sequence of the deduced ATM protein. It has 3056 amino acids. The ATM gene product contains a phosphatidylinositol-3 kinase (PI-3) signature sequence at codons 2855-2875. Mutation analyses in the initial report by Savitsky et al. (K. Savitsky et al. (1995), supra) use restriction endonuclease fingerprinting to identify mutations in the reverse-transcribed 5.9 kb carboxy-terminal end, which included the PI-3 signature sequence, of the 10 kb transcript that was available at that time (K. Savitsky et al., Hum. Mol. Genet. 4:2025-2032 (1995)). Both in-frame and frameshift mutations were found. Because the methodology used for screening for mutations biases the types of mutations found, there is a need to use different screening methods to identify further mutations in the ATM gene. The complete 150 kb genomic sequence was subsequently published (M. Platzer et al., xe2x80x9cAtaxia-Telangiectasia Locus; Sequence Analysis of 184 kb of Human Genomic DNA Containing the entire ATM Gene,xe2x80x9d Genome Res. 7:592-605 91988) and assigned Accession Number V82828.
The ATM gene is an example of a complex polyexonic eukaryotic gene that codes for a large protein product, in which defects appear as autosomal recessive mutations. There exists a large number of clinically important genes of this category, and improved methods of detecting polymorphisms in such genes are needed. In particular, there is a need for methods that can use either DNA or RNA as starting materials so that they are not dependent on existence of RNA molecules. Previous techniques include restriction endonuclease fingerprinting (REF), the single-stranded conformation polymorphism (SSCP) technique and the protein truncation test (PTT). There is also a need for a method that can detect mutations occurring in non-coding regions such as control elements, which would be missed by the protein truncation test. Therefore, there is a need for improved methods of detection of mutations and polymorphisms in such complex polyexonic eukaryotic structural genes.
Because of the severity of the disease associated with mutations in the ATM gene, patients or families frequently request confirmation of a suspected diagnosis of A-T. If the mutation is already known in a family, it is much easier to test other family members to see whether they carry that mutation. Since carriers of ATM mutations (i.e., heterozygotes with one normal gene) may also be at an increased risk of cancer, particularly breast cancer, testing for such mutations has attracted much commercial interest. Automated chips and readers are being developed by many companies; however, these readers have an error rate of about 1/1000, making it difficult to distinguish real mutations from errors or normal variations (i.e., polymorphisms). Approximately 23,000 nucleotides must be screened to identify most ATM mutations. A normal polymorphism appears every 500 nucleotides. Thus, in a region of 23,000 nucleotides being searched, there should be one (or possibly two) mutations amidst 23+46+2=71 errors and polymorphisms. The interpretation of such information is best approached by xe2x80x9clook-upxe2x80x9d tables that list all known polymorphisms and mutations (sometimes referred to as SNPs or single nucleotide polymorphisms. Therefore, there is a need for improved methods of detecting polymorphisms in the ATM gene and in other large, complex, polyexonic genes in order to improve such automated screening.
One aspect of the present invention is method of detecting a mutation or a polymorphism in the human ataxia telangiectasia gene comprising the steps of:
(1) amplifying a plurality of nonoverlapping nucleic acid segments from the human ataxia telangiectasia gene;
(2) subjecting the amplified nonoverlapping nucleic acid segments to single-stranded conformation polymorphism electrophoresis in a number of lanes such that two or three amplified nucleic acid segments are electrophoresed per lane, the electrophoresis of the segments electrophoresed in the same lane being initiated at different times, such that the signals from each amplified nucleic acid segment are distinct in each lane, the time interval between the initiation of electrophoresis for each segment being chosen to ensure that signals resulting from the electrophoresis are distinct for each segment electrophoresed in the same lane; and
(3) comparing the signals from the resulting single-stranded conformation polymorphism electrophoresis for each segment in each of the lanes to detect the mutation or polymorphism.
The plurality of nonoverlapping nucleic acid segments that are amplified can be RNA and the segments can be amplified by the reverse transcriptase-polymerase chain reaction mechanism. Alternatively, the plurality of nonoverlapping nucleic acid segments that amplified can be DNA and the segments can be amplified by the polymerase chain reaction mechanism.
The method preferably further comprises the step of cleaving amplified products larger than about 350 bases with a restriction endonuclease that cleaves the amplified products into fragments that are less than about 350 bases.
Typically, the electrophoresis occurs in polyacrylamide gels with glycerol as a gel matrix from about 150 to about 250 volts for about 14 to 16 hours. Preferably, the electrophoresis is performed in a plurality of gels so that the step of comparing the signals resulting from the electrophoresis of the amplified nucleic acid segments can detect mutations or polymorphisms in a plurality of segments of the gene. This procedure is entitled mega-SSCP.
A set of 70 primers can be used, as shown in Table 1.
The method can also be applied to the detection of mutations or polymorphisms in other genes. These genes include the APC gene, the CFTR gene, the BRCA1 gene, the BRCA2 gene, the HBB gene, the APOE gene, the PRNP gene, the SCA1 gene, the APP gene, the HPRT gene, the PAX3 gene, the RET gene, the PMP22 gene, the SCN4A gene, and the GNAS1 gene.
Another aspect of the present invention is an isolated and purified nucleic acid fragment comprising nucleic acid having complementarity or identity to a mutation in the ataxia-telangiectasia mutated (ATM) gene, the mutation being selected from the group consisting of:
(1) 10744A greater than G;
(2) 11482G greater than A;
(3) IVS3xe2x88x92558A greater than T;
(4) 146C greater than G;
(5) 381delA;
(6) IVS8xe2x88x923delGT
(7) 1028delAAAA
(8) 1120C greater than T;
(9) 1930ins16
(10) IVS16+2T greater than C;
(11) 2572T greater than C;
(12) IVS21+1G greater than A;
(13) 3085delA;
(14) 3381delTGAC;
(15) 3602delTT;
(16) 4052delT;
(17) 4396C greater than T;
(18) 5188C greater than T;
(19) 5290delC;
(20) 5546delT;
(21) 5791G greater than CCT;
(22) 6047A greater than G;
(23) IVS44xe2x88x921G greater than T;
(24) 6672delGC/6677delTACG;
(25) 6736del11/6749del7;
(26) 7159insAGCC;
(27) 7671delGTTT;
(28) 7705del14
(29) 7865C greater than T;
(30) 7979delTGT;
(31) 8177C greater than T;
(32) 8545C greater than T;
(33) 8565T greater than A;
(34) IVS64+IG greater than T; and
(35) 9010 del28.
Yet another aspect of the present invention is an isolated and purified nucleic acid fragment comprising nucleic acid having complementarity or identity to a polymorphism or SNP in the ataxia-telangiectasia mutated (ATM) gene, the polymorphism being selected from the group consisting of:
(1) 10807A greater than G;
(2) IVS3xe2x88x92122T greater than C;
(3) IVS6+70delT;
(4) IVS 1 6xe2x88x9234C greater than A;
(5) IVS22xe2x88x9277T greater than C;
(6) IVS24xe2x88x929delT;
(7) IVS25xe2x88x9213delA;
(8) 5557G greater than A;
(9) IVS48xe2x88x9269insATT; and
(10) IVS62xe2x88x9255T greater than C.
These polymorphisms are relatively common polymorphisms.
Yet another aspect of the present invention is an isolated and purified nucleic acid fragment comprising nucleic acid having complementarity or identity to a polymorphism in the ataxia-telangiectasia mutated (ATM) gene, the polymorphism being selected from the group consisting of:
(1) 10677G greater than C;
(2) 10742G greater than T;
(3) 10819G greater than T;
(4) 10948A greater than G;
(5) IVS3xe2x88x92300G greater than A;
(6) IVS8xe2x88x9224del5;
(7) IVS13xe2x88x92137T greater than C;
(8) IVS14xe2x88x9255T greater than G;
(9) 1986T greater than C;
(10) IVS20+27delT;
(11) IVS23xe2x88x9276T greater than C;
(12) IVS25xe2x88x9235T greater than A;
(13) IVS27xe2x88x9265T greater than C;
(14) IVS30xe2x88x9254T greater than C;
(15) 4362A greater than C;
(16) IVS38xe2x88x928T greater than C;
(17) 5793T greater than C;
(18) IVS47xe2x88x9211G greater than T;
(19) IVS49xe2x88x9216T greater than A;
(20) IVS53+34insA;
(21) IVS60xe2x88x9250delTTAGTT;
(22) IVS62+8A greater than C;
(23) IVS62xe2x88x9265G greater than A; and
(24) 9200C greater than G.
These are relatively rare polymorphisms.
Another aspect of the present invention is a method for testing a DNA sample of a human for the presence or absence of a mutation or polymorphism in the ATM gene comprising the steps of:
(1) providing a sample of DNA from a human; and
(2) testing the sample for the presence of a mutation or a polymorphism in the ATM gene, the mutation or the polymorphism being one of the mutations or polymorphisms described above.
Yet another aspect of the present invention is an isolated and purified protein, polypeptide, or peptide encoded by a polynucleotide that comprises one of the fragments described above.
Still another aspect of the present invention is an antibody that specifically binds the isolated and purified protein, polypeptide, or peptide.
Another aspect of the present invention is a transgenic mammal all of whose germ cells and somatic cells contain the fragment described above introduced into the mammal or an ancestor of the mammal at an embryonic stage. Typically, the transgenic mammal is a mouse.